Multiphoton microscopy imaging: ntau hom kev yees duab rau cov neurons hauv vivo
Piv nrog rau cov tshuaj ib leeg-photon wide-field fluorescence microscope, multiphoton microscopy (MPM) muaj lub luag haujlwm ntawm optical sectioning thiab sib sib zog nqus duab. Hauv 2019, Jerome Lecoq et al. tham txog MPM thev naus laus zis los ntawm peb yam: neuron imaging tob hauv lub hlwb, loj neuron imaging, thiab high-speed neuron imaging.
Txhawm rau txuas cov kev ua haujlwm neuron nrog cov cwj pwm nyuaj, feem ntau nws yog qhov tsim nyog rau cov duab neurons nyob rau hauv qhov tob cortex, uas yuav tsum tau MPM kom muaj peev xwm ntawm cov duab sib sib zog nqus. Excitation thiab emission teeb yuav tawg heev thiab absorbed los ntawm cov ntaub so ntswg lom, uas yog lub ntsiab tseem ceeb txwv qhov tob ntawm MPM. Txawm hais tias qhov teeb meem tawg tuaj yeem daws tau los ntawm kev ua kom lub zog ntawm laser, nws yuav ua rau muaj lwm yam teeb meem, xws li hlawv cov qauv, defocusing thiab ze-nto Fluorescent excitation. Txoj hauv kev zoo tshaj plaws los ua kom qhov tob ntawm MPM duab yog siv cov wavelengths ntev dua li lub teeb pom kev zoo.
Tsis tas li ntawd, rau ob-photon (2P) imaging, out-of-focus thiab ze-surface fluorescence excitation yog ob qhov loj tshaj plaws qhov tob-limiting yam, thaum rau peb-photon (3P) imaging, ob qhov teeb meem no yog heev txo, tab sis Peb-photon imaging vim fluorescence Kev nqus ntawm ntu ntu ntawm pab pawg tsawg dua li ntawm 2P, yog li qhov kev txiav txim siab siab dua lub zog mem tes yuav tsum tau txais tib lub zog fluorescence teeb liab uas zoo siab los ntawm 2P. Functional 3P microscopy yog qhov xav tau ntau dua li cov qauv 3P microscopy, uas yuav tsum tau kuaj xyuas sai dua thiaj li ua rau cov kev ua haujlwm neuronal hauv lub sijhawm; Lub zog pulse ntau dua yuav tsum tau txhawm rau txhawm rau sau cov cim txaus hauv lub sijhawm nyob ntawm txhua pixel.
Cov cwj pwm nyuaj feem ntau cuam tshuam nrog lub hlwb loj nrog kev sib txuas hauv zos thiab ntev. Txhawm rau txuas cov haujlwm neuron nrog tus cwj pwm, nws yog ib qho tsim nyog los saib xyuas cov haujlwm ntawm cov neurons loj heev thiab nthuav dav nyob rau tib lub sijhawm. Lub neural network hauv lub hlwb ua cov khoom stimuli hauv kaum tawm milliseconds. Txhawm rau kom nkag siab qhov ceev ceev neural network Los kawm txog cov neuron dynamics, MPM yuav tsum muaj peev xwm ua kom pom cov duab neurons sai. Txoj kev ceev MPM tuaj yeem muab faib ua cov txheej txheem ib leeg-nqaj scanning thiab ntau-beam scanning cov txheej txheem.
Tib-beam scanning thev naus laus zis ua rau muaj kev kub ceev traversal ntawm neural cov ntaub so ntswg nrog kev pom loj (FOV)
Thaum siv MPM rau cov duab neurons, random-access scanning-uas yog, lub laser beam yog scanned sai sai ntawm txhua qhov chaw xaiv ntawm tag nrho cov kev pom - tuaj yeem luam theej duab tsuas yog cov paj hlwb ntawm kev txaus siab, uas tsis tsuas yog zam kev luam tawm ib qho tsis muaj cov paj hlwb tuaj yeem. kuj optimize lub sij hawm scanning ntawm lub laser beam. Random-access scanning (Daim duab 1) tuaj yeem ua tiav nrog acousto-optic deflector (AOD), uas ua haujlwm los ntawm kev sib txuas lub piezoelectric transducer nrog lub xov tooj cua zaus teeb liab mus rau ib qho khoom siv lead ua. Qhov tshwm sim acoustic nthwv dej ua rau lub sijhawm ncua sij hawm refractive index grating, Diffraction tshwm sim thaum lub laser beam hla los ntawm grating. Qhov kev siv thiab zaus ntawm lub suab nthwv dej tuaj yeem hloov kho los ntawm lub xov tooj cua zaus hluav taws xob teeb liab los hloov qhov kev siv thiab kev coj ntawm lub teeb diffracted, kom ib sab kab rov tav taw tes scanning tuaj yeem pom tau los ntawm kev siv ib qho AOD, thiab 3D tuaj yeem pom tau. los ntawm kev siv ib khub ntawm AODs ua ke nrog rau lwm yam axial scanning technologies random access scanning. Txawm li cas los xij, cov txheej txheem no yog rhiab heev rau cov lus tsa suab ntawm cov qauv thiab nquag mus rau cov khoom siv dag zog. Tam sim no, ceev raster scanning, uas yog, nce qib scanning nyob rau hauv FOV, yog dav siv vim hais tias cov algorithm yuav yooj yim daws tau cov duab artifacts.
AOD-raws li ob-photon duab ntawm neocortical L2/3 neurons hauv vivo[2]
Muaj ntau txoj hauv kev kom paub ceev raster scanning, siv daim iav vibrating rau ceev 2D scanning, combining daim iav vibrating thiab adjustable fais lens rau ceev 3D scanning, tab sis lub adjustable hluav taws xob lens tsis tau ceev ceev ceev nyob rau hauv lub axial kev taw qhia vim muaj kev txwv. mechanical inertia Switching, uas cuam tshuam rau cov duab nrawm, tam sim no tuaj yeem hloov nrog lub teeb pom kev zoo (SLM).
Kev tsom xam tej thaj chaw deb kuj yog ib qho txhais tau tias kom ua tiav 3D duab, raws li qhia hauv daim duab 2. Hauv LSU module, lub scanning galvanometer scans kab rov tav, thiab ASU module suav nrog lub hom phiaj lens L1 thiab daim iav M, thiab axial scanning yog pom tau los ntawm kev kho. txoj hauj lwm ntawm M. Cov txheej txheem no tuaj yeem tsis tsuas yog kho qhov kho qhov muag aberration qhia los ntawm lub hom phiaj tseem ceeb L2, tab sis kuj pab kom ceev axial scanning. Txhawm rau kom tau txais ntau cov duab neuron, FOV tuaj yeem nthuav dav los ntawm kev kho lub hom phiaj lens tsim ntawm lub tshuab tsom, tab sis lub hom phiaj lens nrog loj NA thiab loj FOV feem ntau hnyav thiab tsis tuaj yeem txav mus sai rau kev soj ntsuam axial ceev, yog li FOV loj loj cia siab rau Telefocus. , SLM thiab adjustable motorized lo ntsiab muag.
Schematic daim duab ntawm lub chaw taws teeb tsom rau ob-photon imaging system[3] Multi-beam scanning thev naus laus zis tuaj yeem ua duab sib txawv ntawm cov ntaub so ntswg neuronal.
This technique3 typically uses two independent paths for imaging two distant (>1-2 hli sib nrug) qhov chaw duab (Fig. 3C,D); rau cov cheeb tsam uas nyob ib sab, nws feem ntau siv ntau kab ntawm ib lub hom phiaj lens rau kev yees duab (Fig. 3E, F). Cov txheej txheem multi-beam scanning yuav tsum tau them nyiaj tshwj xeeb rau cov teeb meem crosstalk ntawm excitation beams, uas tuaj yeem daws tau los ntawm kev sib cais tom qab lub teeb los yog qhov chaw-lub sij hawm multiplexing txoj kev. Txoj kev sib cais ntawm lub teeb tom qab hoc yog hais txog kev siv cov algorithms los cais cov kab teeb kom tshem tawm cov crosstalk; lub sij hawm-qhov chaw multiplexing txoj kev yog hais txog kev siv ib txhij ntawm ntau txoj kab hluav taws xob excitation, lub pulses ntawm txhua lub beam yog ncua sij hawm, kom tus neeg beams zoo siab los ntawm cov kab teeb sib txawv tuaj yeem sib cais ib ntus. fluorescent teeb liab. Ntau cov neurons tuaj yeem ua duab los ntawm kev nthuav qhia ntau kab teeb, tab sis ntau kab teeb yuav ua rau kom qhov sib tshooj ntawm fluorescence lwj lub sij hawm, uas txwv tsis pub muaj peev xwm paub qhov txawv ntawm cov teeb liab; thiab multiplexing muaj kev cuam tshuam tsis zoo rau kev ua haujlwm ceev ntawm cov khoom siv hluav taws xob. Kev xav tau siab; ntau cov kab teeb kuj yuav tsum muaj lub zog laser ntau dua los tswj qhov kwv yees ntawm cov teeb liab-rau-nruab nrab ntawm ib kab teeb, uas tuaj yeem yooj yim ua rau cov ntaub so ntswg puas.
Loj cheeb tsam imaging Technology
Nyob rau hauv xyoo tas los no, kev txhim kho ntawm MPM technologies sib txawv tau nthuav dav ntawm peb cov duab ntawm cov ntaub so ntswg neural, tso cai rau peb kom pom ntau cov neurons sib sib zog nqus hauv lub hlwb ntawm qhov nrawm nrawm, uas tau txhawb nqa kev tshawb fawb neuroscience zoo heev thiab ua rau peb tau txais kev nkag siab zoo dua. ntawm lub hlwb ua haujlwm.
